Mitochondrial dysfunction has been implicated in the etiology and expression of insulin resistance and type 2 diabetes in animal models and humans under conditions of high fat loads either from the diet, endogenous release by adipose tissue, or both. However, controversy exists as to whether insulin resistance under these conditions results from intrinsic defects in mitochondrial energy utilization or abnormalities in free fatty aid (FFA) flux and amino acid metabolism, including the potential role of accumulated metabolic intermediates to impair insulin signaling. To address these controversies, we propose two aims that will formally test the hypothesis that intrinsic defects in mitochondrial function involving long-chain, but not medium-chain, FAO will prevent intralipid-induced insulin resistance using advanced metabolomics methodology in combination with hyperinsulinemic-euglycemic clamps and analyses of insulin signaling in muscle and fat biopsies. Study subjects will be recruited from a unique population of patients with inherited defects in one of three mitochondrial enzymes in the fatty acid oxidation pathway (FAO), including oxidation of long-chain fatty acids (very long-chain acylCoA dehydrogenase (VLCAD), or trifunctional protein (TFP), including long-chain 3-hydroxy acylCoA dehydrogenase (LCHAD) deficiencies), and, finally, medium-chain fatty acids (medium-chain acyl CoA dehydrogenase, or MCAD). These patients and age, sex, and BMI-matched healthy controls will undergo testing on separate visit study dates during which they receive co-infusions of either intralipid, shown to induce ectopic lipid accumulation and insulin resistance in previous studies, or a control infusion of glycerol and saline. These detailed studies in patients with FAO disorders compared to controls will give us a unique opportunity to better understand the interface between fatty acid metabolism and regulation of insulin sensitivity in humans. Not only will these studies address current controversies and gaps in our understanding in this field, but by including measurements of both cellular and systemic metabolomics, they may also help inform on-going development of potential pharmaceutical targets for the treatment of insulin resistance and type 2 diabetes.